Method for providing neuroprotection from spinal muscular atrophy

ABSTRACT

A method for providing neuroprotection to a patient in need of neuroprotection, comprising administering a neuroprotective-effective amount of a compound of formula I 
     
       
         
         
             
             
         
       
         
         
           
             in which X is an oxygen atom or an ═N—OH group, 
             R is selected from the group consisting of 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             A is a hydrogen atom or together with B a carbon-carbon bond, 
             B is a hydrogen atom, a hydroxy group or together with A a carbon-carbon bond, 
             C is a hydrogen atom or together with D a carbon-carbon bond, 
             D is a hydrogen atom or together with C a carbon-carbon bond, 
             E is a hydrogen atom or together with F a carbon-carbon bond, 
             F is a hydrogen atom or together with E a carbon-carbon bond, 
             or an addition salt with a pharmaceutically acceptable acid.

The present invention relates to the use as medicaments of derivativesof cholest-4-en-3-one, in particular as neuroprotectors for example inthe pathologies and the traumatisms linked to the degeneration or deathof the motor neurons, the pharmaceutical compositions containing them,novel derivatives and their preparation process.

Neurodegenerative processes are characterized by the dysfunction anddeath of the neurons leading to the loss of the neurological functionsmediated by the brain (central nervous system, CNS), the spinal cord andthe peripheral nervous system (PNS). They can result, amongst others,from pathological situations known collectively under the term ofneurodegenerative diseases or affections, traumatism, or exposure totoxins.

The most important pathologies which are characterized by a degenerativeprocess are:

-   -   the hereditary or sporadic, chronic neurodegenerative diseases,        in particular Alzheimer's disease, Huntington's chorea,        Parkinson's disease, amyotrophic lateral sclerosis, spinal        amyotrophies, Creutzfeldt-Jakob's disease, multiple sclerosis,        adrenoleukodystrophy, epilepsy, dementia, schizophrenia, and the        neurological syndromes associated with AIDS;    -   neuronal lesions linked with ageing;    -   the hereditary peripheral neuropathies or peripheral        neuropathies resulting from a lesion, such as Fabry's disease,        Charcot-Marie-Tooth disease, Krabbe's disease, leukodystrophies,        diabetic neuropathies and those induced by anti-cancer        treatments;    -   traumatisms of the brain, the peripheral nerves or the spinal        cord;    -   ischemias of the brain or the spinal cord following a        cerebro-vascular accident, or induced by a lack of blood        irrigation;    -   degenerations which are hereditary, traumatic or linked with        ageing of the sensory neurons of vision, such as the macular        degenerations, pigmentary retinitis, or degenerations of the        optic nerve induced by glaucomas;    -   degenerations which are hereditary, resulting from a lesion or        linked with ageing of the sensory neurons of hearing leading to        a reduction or a loss of audition.

A part of the signalling pathways affected in these pathologies arecommon to a large number of neurodegenerative diseases. Alzheimer'sdisease is the most frequent dementia. It causes the appearance ofatrophy of the brain, a predominant neuronal loss in the hippocampus andit also affects the cholinergic neurons. Other pathologies, such as thelobar atrophies (Pick's disease, Creutzfeld-Jakob's disease), Lewy bodydementia, vascular dementias, Parkinson's disease are associated withsignificant neuronal death at the outset of the symptoms of thesedementias.

Currently an effective treatment does not exist to check the neuronaldegenerations. A therapeutic approach for protecting the neurons fromdying is the supply of neurotrophic proteins.

These proteins, such BDNF (brain-derived neurotrophic factor), CNTF(ciliary neurotrophic factor), NGF (nerve growth factor), GDNF(glia-derived neurotrophic factor) are synthesized during embryonaldevelopment or after lesion in adults. These growth factors encouragethe survival, maturation and differentiation of neuronal cells.Moreover, they inhibit the apoptotic mechanisms, activate multiplesurvival pathways and protect a large number of neuronal populations.Their use is proposed in the majority of the neuronal degenerations.

Compounds which would activate the expression of neurotrophic factors orwhich would mimic the action of these factors have a therapeuticpotential for the treatment of neurodegenerative syndromes.

In particular, the supply of neurotrophic molecules for the treatment ofneuronal degenerations has three objectives:

-   -   to compensate for a potential deficiency in neurotrophic factors        linked to a failure of supply by the peripheral or central        targets of the neurons and/or a problem with the retrograde        transport of these factors;    -   to intervene in a non-specific fashion on the biochemical        pathways involved in the degenerative cascade;    -   to encourage the natural compensator phenomena of dendritic        growth and arborization of the nerve endings.

These compounds would therefore have a beneficial effect in a largenumber of pathologies in particular in the pathologies affecting theperipheral and central nervous systems.

Moreover, within the above context, the motor neurons are neurons inparticular present in the spinal cord and the brain stem. Theirdegeneration or their death can lead to a progressive weakening of themuscles of the limbs, then to atrophy and possibly to spasticity (i.e. apermanent contraction) of the muscle.

The most important pathologies which result from the degeneration anddeath of the spinal and/or bulbar motor neurons are amyotrophic lateralsclerosis, also known under the name of Charcot's disease or also LouGehrig's disease, and infantile spinal amyotrophies, also known underthe names of Werdnig-Hoffmann disease or Kugelberg-Welander disease.

Moreover, a degeneration of the motor neurons is observed in the case oftraumas with crushing and/or severing of the spinal cord or theperipheral motor nerves.

More generally, spinal amyotrophies are referred to as diseases wherethe degeneration or death of the motor neurons of the spinal cord areinvolved.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative diseaseassociated with different types of inclusions such as Lewy bodies andcharacterized by a degeneration of the spinal and cortical motor neuronsthe fatal outcome of which is sometimes associated with frontaldementia. During the development of ALS, the degenerative phenomenaoccur not only in the brain but also in the spinal cord and consequentlyin the muscle, by lack of innervation.

Active compounds for combating the diseases mentioned above are alwaysbeing sought.

Now the Applicant has discovered that the derivatives of4-cholesten-3-one and in particular cholest-4-en-3-one oxime are endowedwith remarkable neuroprotective properties, particularly vis à vis themotor neurons, the neurons of the central nervous system, of the motorand peripheral nerves, and therefore are of use as medicaments, and thatcertain of them are moreover endowed with remarkable properties forinhibiting the effect of positive allosteric modulators of the GABA_(A)(gamma amino butyric acid of type A) receptors.

This is why a subject of the present invention is the compoundscorresponding to the formula I

in which X represents an oxygen atom or an ═N—OH group,

R represents a group chosen from

A represents a hydrogen atom or together with B a carbon-carbon bond,

B represents a hydrogen atom, a hydroxy group or together with A acarbon-carbon bond,

C represents a hydrogen atom or together with D a carbon-carbon bond,

D represents a hydrogen atom or together with C a carbon-carbon bond,

E represents a hydrogen atom or together with F a carbon-carbon bond,

F represents a hydrogen atom or together with E a carbon-carbon bond,

as well as their addition salts with pharmaceutically acceptable acids,with the exception of cholest-4-en-3-one,24-ethylcholest-4,22-dien-3-one, 5-beta-cholestan-3-one andcholest-4,6-dien-3-one as well as cholest-4,24-dien-3-one,24-methylcholest-4,6,22-trien-3-one, and cholest-5-en-3-one oxime, fortheir use in a therapeutic treatment method for the human or animalbody, i.e. as medicaments.

The addition salts with pharmaceutically acceptable acids can be forexample the salts formed with the following acids: hydrochloric,hydrobromic, nitric, sulphuric, phosphoric acetic, formic, propionic,benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, glyoxylic,aspartic, alkane sulphonic such as methane or ethane sulphonic acids,arylsulphonic, such as benzene or paratoluene sulphonic acids, orcarboxylic acid.

Among the compounds described above, the compounds of formula I forwhich X represents an ═N—OH group as well as their addition salts withpharmaceutically acceptable acids are in particular retained.

More particularly the above compounds are retained for which,

-   -   X represents an ═N—OH group, A represents together with B a        carbon-carbon bond, C, D, represent a hydrogen atom, E, F        represent a hydrogen atom or together a carbon-carbon bond and R        has the meaning R1,    -   X represents an ═N—OH group, A represents together with B a        carbon-carbon bond, C, D represent a hydrogen atom, E, F        represent a hydrogen atom and R has the meaning R2 or R3 or R4,    -   X represents an ═N—OH group, A represents together with B a        double bond, C represents together with D a carbon-carbon bond,        E, F represent a hydrogen atom and R has the meaning R1 or R6,    -   X represents an ═N—OH group, A represents together with B a        double bond, C represents together with D a carbon-carbon bond,        E represents together with F a carbon-carbon bond represent a        hydrogen atom and R has the meaning R1    -   X represents an ═N—OH group, E represents together with F a        double bond, C, D, A, B represent a hydrogen atom and R has the        meaning R1, as well as their addition salts with        pharmaceutically acceptable acids

Among the compounds of the invention there can moreover be mentioned

-   -   5β-hydroxy-cholest-4-en-3-one    -   cholestan-3-one oxime        as well as their addition salts with the pharmaceutically        acceptable acids.

Quite particularly the following are retained:

-   -   cholest-4-en-3-one oxime,    -   1,4-cholestadien-3-one oxime        as well as their addition salts with the pharmaceutically        acceptable acids.

The compounds which are a subject of the present invention have veryuseful pharmacological properties. They are endowed in particular withremarkable neuroprotective properties, particularly vis à vis the motorneurons.

These properties are illustrated hereafter in the experimental part.They justify the use of the compounds described above as well as theiraddition salts with the pharmaceutically acceptable acids as amedicament.

The medicaments according to the present invention are of use due totheir neuroprotectrive properties for example in the treatment or theprevention of the neurodegenerative diseases, such as for exampleHuntington's disease, hereditary or sporadic, chronic neurodegenerativediseases, neuronal lesions linked with ageing, hereditary peripheralneuropathies or peripheral neuropathies resulting from a lesion,Charcot-Marie-Tooth disease, diabetic neuropathies or neuropathiesinduced by anti-cancer treatments, traumas of the brain, of theperipheral nerves or of the spinal cord, ischemias of the brain or thespinal cord, degenerations which are hereditary, resulting from a lesionor linked with ageing of the sensory neurons of vision or degenerationsof the optic nerve, degenerations which are hereditary, traumatic orlinked with ageing of the sensory neurons of hearing, lobar atrophiesand vascular dementias, and in particular spinal amyotrophies,amyotrophic lateral sclerosis and pathologies due to traumas of thespinal cord or the peripheral motor nerves.

In the context of the invention, the term “treatment” designatespreventive, curative, palliative treatment, as well as caring forpatients (reduction of suffering, improvement in life span, slowing downthe progression of the disease), etc. The treatment can moreover becarried out in combination with other ingredients or treatments, such asin particular other active compounds for treating the pathologies ortraumatisms specified in the present Application.

The subject components also have properties which inhibit the effect ofpositive allosteric modulators of the GABA_(A) (gamma amino butyric acidof type A) receptors. Examples of positive allosteric modulators are:allopregnanolone or 3α-hydroxy-5α-pregnan-20-one or 3α,5α-TH-PROG ortetrahydrodeoxycorticosterone=3α,20-dihydroxy-5α-pregnan-20-one=3α,5α-TH-DOC,which are endogenous neurosteroids of the central nervous system. Thesespecific properties justify that the compounds of the invention can alsobe of use in the treatment of different physiopathological situationswhere these neurosteroids have been described as playing an importantrole, such as for example,

-   -   sexual behaviour    -   sudden death of the newborn, and in particular    -   painful sensitivity or chronic neuropathic pain    -   anxiety or depression    -   traumatic lesions of the nervous system    -   steroid-sensitive epilepsy, sleep disorders or alcohol        intoxication    -   cognitive learning or memory disorders.

In particular, due to their neuroprotective properties vis à vis themotor neurons, they are of particular use in the treatment ofamyotrophies, in particular amyotrophic lateral sclerosis or infantilespinal muscular atrophy, and in the treatment of traumas of the spinalcord or the peripheral motor nerves as mentioned above.

In general the daily dose of compound is the minimum dose to obtain thetherapeutic effect. This dose will depend on the different factorsmentioned previously. The doses of the compounds described above and forexample cholest-4-en-3-one oxime are in general comprised between 0.001to 100 mg per kilo per day for man.

If necessary, the daily dose can be administered in two, three, four,five, six or more, doses per day or by multiple sub-doses administeredat appropriate intervals during the day.

The quantity chosen will depend on multiple factors, in particular theadministration route, the duration of administration, the time ofadministration, the speed of elimination of the compound, the differentproduct(s) used in combination with the compound, the age, weight andphysical condition of the patient, as well as the patient's medicalhistory, and all other information known in medicine.

The prescription of the attending physician can start at doses lowerthan those generally used, then these doses will be progressivelyincreased in order to better control the appearance of possible sideeffects.

A subject of the invention is also the pharmaceutical compositions whichcontain at least one above-mentioned compound or one of its additionsalts with pharmaceutically acceptable acids, as active ingredient.

In these compositions, the active ingredient is advantageously presentat physiologically effective doses; the above-mentioned compositionscontain, in particular, an effective neuroprotectrive dose of at leastone of the above active ingredients.

As medicaments, the compounds corresponding to formula I as well astheir addition salts with pharmaceutically acceptable acids can beincorporated in pharmaceutical compositions intended for the digestiveor parenteral route.

The pharmaceutical compositions according to the invention can comprisemoreover at least one other therapeutically active ingredient, for usewhich is simultaneous, separated or spread over time, in particularduring a treatment for a subject suffering from a pathology or a traumalinked to the degeneration or to death of the motor neurons as definedabove.

The pharmaceutical compositions or medicaments according to theinvention advantageously include one or more inert excipients orvehicles, i.e. pharmaceutically inactive and non toxic. There can bementioned for example saline, physiological, isotonic, bufferedsolutions, etc., compatible with pharmaceutical use and known to aperson skilled in the art. The compositions can contain one or moreagents or vehicles chosen from dispersants, solubilizing agents,stabilizers, preservatives, etc. Agents or vehicles which can be used informulations (liquids and/or injectable solutions and/or solids) are inparticular methylcellulose, hydroxymethylcellulose,carboxymethylcellulose, cyclodextrins, polysorbate 80, mannitol,gelatin, lactose, vegetable or animal oils, acacia, etc. Thecompositions can be formulated in the form of injectable suspensions,gels, oils, tablets, suppositories, powders, gelatin capsules, capsules,etc., optionally using galenic forms or devices ensuring a sustainedand/or controlled release. For this type of formulation, an agent suchas cellulose, carbonates or starches is advantageously used.

Administration can be carried out by any method known to a personskilled in the art, preferably by oral route or by injection, typicallyby intra-peritoneal, intra-cerebral, intra-thecal, intravenous,intra-arterial or intra-muscular route. Administration by oral route ispreferred. If it is a question of a long term treatment, the preferredadministration route is sublingual, oral or transcutaneous.

For injections, the compounds are generally packaged in the form ofliquid suspensions, which can be injected using syringes or perfusions,for example.

It is understood that the flow rate and/or the dose injected orgenerally the dose to be administered, can be adapted by a personskilled in the art as a function of the patient, the pathology,administration method, etc. It is understood that the repeatedadministrations can be carried out, optionally in combination with otheractive ingredients or any vehicle which is acceptable on thepharmaceutical level (buffers, saline, isotonic solutions, in thepresence of stabilizing agents, etc.).

The invention can be used in mammals, in particular in humans.

A subject of the present invention is also a process for the preparationof a composition described above, characterized in that the activeingredient or ingredients are mixed, according to the methods known perse, with acceptable excipients, in particular pharmaceuticallyacceptable.

The compounds of formula I as defined above are known or can be preparedaccording to the processes described in the literature. Certainderivatives of formula I are novel products.

This is why a subject of the present invention is also the novelcompounds corresponding to formula I

in which X represents an ═NH—OH radical and

-   -   A represents together with B a carbon-carbon bond, C, D        represent a hydrogen atom, E represents together with F a        carbon-carbon bond and R has the meaning R1,    -   A represents together with B a carbon-carbon bond, C, D        represent a hydrogen atom, E, F represent a hydrogen atom and R        has the meaning R2 or R3 or R4,    -   A represents together with B a carbon-carbon bond, C represents        together with D a carbon-carbon bond, E, F represent a hydrogen        atom and R has the meaning R6,    -   A represents together with B a carbon-carbon bond, C represents        together with D a carbon-carbon bond, E represents together with        F a carbon-carbon bond and R has the meaning R1,        as well as their addition salts with the mineral or organic        acids.

A subject of the present invention is also a process for the preparationof novel compounds of formula I as defined above as well as their salts,characterized in that a compound of formula II

in which

-   -   A represents together with B a carbon-carbon bond, C, D        represent a hydrogen atom, E represents together with F a        carbon-carbon bond and R has the meaning R1,    -   A represents together with B a carbon-carbon bond, C, D        represent a hydrogen atom, E, F represent a hydrogen atom and R        has the meaning R2 or R3 or R4,    -   A represents together with B a carbon-carbon bond, C represents        together with D a carbon-carbon bond, E, F represent a hydrogen        atom and R has the meaning R6,    -   A represents together with B a carbon-carbon bond, C represents        together with D a carbon-carbon bond, E represents together with        F a carbon-carbon bond and R has the meaning R1,

is reacted with a hydroxylamine halide such as hydroxylaminehydrochloride, in order to obtain the expected compound of formula Iwhich is isolated and if desired salified.

Under preferential conditions for implementing the process describedabove,

-   -   the starting product is solubilized in a minimum amount of a        suitable solvent such as pyridine    -   if another ketone function is present, it is specifically        blocked by a suitable protective group such as cyclic acetals,    -   an excess, for example 2 equivalents, of hydroxylamine halide is        used.    -   the operation is carried out under stirring for approximately 24        hours at ambient temperature.

The compounds of formula II are known derivatives, described in theliterature, and are commercially accessible.

A subject of the invention is also the use of a compound of formula I

in which X represents an oxygen atom or an ═N—OH group,

R represents a group chosen from

A represents a hydrogen atom or together with B a carbon-carbon bond

B represents a hydrogen atom, a hydroxy group or together with A acarbon-carbon bond,

C represents a hydrogen atom or together with D a carbon-carbon bond,

D represents a hydrogen atom or together with C a carbon-carbon bond,

E represents a hydrogen atom or together with F a carbon-carbon bond,

F represents a hydrogen atom or together with E a carbon-carbon bond,

as well as cholest-4,24-dien-3-one, 24-methylcholest-4,6,22-trien-3-one,or cholest-5-en-3-one oxime or one of its addition salts withpharmaceutically acceptable acids, for obtaining a neuroprotectivemedicament, in particular intended for the treatment ofneurodegenerative diseases such as for example Huntington's disease,hereditary or sporadic, chronic neurodegenerative diseases, neuronallesions linked with ageing, hereditary peripheral neuropathies orperipheral neuropathies resulting from a lesion, Charcot-Marie-Toothdisease, diabetic neuropathies or neuropathies induced by anti-cancertreatments, traumas of the brain, of the peripheral nerves or of thespinal cord, ischemias of the brain or the spinal cord, degenerationswhich are hereditary, resulting from a lesion or linked with ageing ofthe sensory neurons of vision or degenerations of the optic nerve,degenerations which are hereditary, traumatic or linked with ageing ofthe sensory neurons of hearing, lobar atrophies and vascular dementias,the diseases and traumas linked to the degeneration of motor neurons andmore particularly the spinal amyotrophies particularly infantile,amyotrophic lateral sclerosis, multiple sclerosis and the traumas of thespinal cord or the peripheral motor nerves.

A particular subject of the invention is the use of a compound offormula I above for obtaining a neuroprotective medicament, inparticular intended for the treatment of pathologies or traumas linkedto the degeneration or death of the neurons, in mammals (in generalpatients) suffering from such pathologies or traumatisms.

A more particular subject of the invention is the use of a compound offormula I for obtaining a medicament intended for the treatment ofinfantile spinal muscular atrophy and amyotrophic lateral sclerosis.

A subject of the invention is also the use of a compound of formula Iabove for obtaining a medicament intended for the treatment of thepathologies where the overactivation of the GABA_(A) receptors, (forexample as a result of the presence of neurosteroids such asallopregnanolone and/or tetrahydrodeoxycorticosterone), can have aharmful effect such as steroid-sensitive epilepsies, alcoholintoxication, cognitive learning, painful sensitivity or sleepdisorders. This overactivation of the GABA_(A) receptors, can becharacterized by the inhibitory effects or the stimulating effects.

The use of these medicaments usually comprises the administration tothese mammals of a therapeutically effective quantity of a compound offormula I and in particular cholest-4-en-3-one oxime, in particular forincreasing the survival of the neurons or encouraging axonal growth.

The invention just as much relates to a treatment method for diseases,in particular neurodegenerative, mentioned above and in particular atreatment method for pathologies or traumas linked to the degenerationor death of the neurons, in mammals (in general patients) suffering fromsuch pathologies or traumas, comprising the administration to thesemammals of a therapeutically effective quantity of cholest-4-en-3-oneoxime, in particular for increasing the survival of the neurons orencouraging axonal growth.

In addition a subject of the invention is a treatment method for one ofthe diseases described above and in particular the pathologies ortraumas linked to the degeneration or death of the motor neurons, inmammals (in general patients) suffering from such pathologies ortraumas, comprising the administration to these mammals of atherapeutically effective quantity of a compound of formula I, inparticular for increasing the survival of the neurones. Morespecifically, the pathologies linked to the degeneration or death of themotor neurons are amyotrophic lateral sclerosis or infantile spinalamyotrophies.

The invention relates both to the provision of novel derivatives of4-cholesten-3-one and to derivatives of 4-cholesten-3-one other thanthose which may have been described in the state of the art. Thosedescribed in the literature are therefore excluded.

The preferential conditions described above for the use of themedicaments of formula I also apply to the other subjects of theinvention mentioned above, in particular to compositions, novelderivatives, uses and treatment methods and vice versa.

The examples which follow illustrate the present application.

EXAMPLE 1

A suspension was prepared corresponding to the formula

Cholest-4-en-3-one oxime 20 mg per ml Excipient: Oily emulsion

EXAMPLE 2

Soft gelatin capsules were prepared corresponding to the formula

Cholest-4-en-3-one oxime 250 mg Excipient: sufficient quantity for agelatin capsule completed at 750 mg

EXAMPLE 3 1,4-cholestadien-3-one oxime (R=R1)

5 g of 1,4-cholestadien-3-one (13 mmol) is solubilized in 50 ml ofpyridine in a 100 ml flask, then 5 g of hydroxylamine hydrochloride isadded. Stirring is maintained for 24 hours at ambient temperature, andthe solvent is evaporated off under reduced pressure. Water then ethylacetate are added in order to carry out an extraction. The organic phaseis washed with an acidified aqueous solution (HCl 1%). The ethyl acetateis evaporated off under reduced pressure. A white powder is obtainedwith a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass spectrometry (Electrospray®)    -   Conditions of the high-performance liquid chromatography:

Column: Macherey-Nagel—Nucleosil® 300-6 C4—150×4.6 mm

Gradient: water (+0.05% TFA)/acetonitrile (+0.05% trifluoroacetic acid)

-   -   t=0 min: 60% acetonitrile, 40% H₂O    -   t=6 min: 100% acetonitrile, 0% H₂O

Then 100% acetonitrile for 5 min.

Retention time: 5 min 60 seconds

Peak detected by mass spectrometry: {M+H}⁺=398

EXAMPLE 4 4,24-cholestadien-3-one oxime (R=R 2)

100 mg of 4,24-cholestadien-3-one (0.26 mmol) is solubilized in 5 ml ofpyridine in a 10 ml flask, then 100 mg of hydroxylamine hydrochloride isadded. Stirring is maintained for 24 hours at ambient temperature, andthe solvent is evaporated off under reduced pressure. Water then ethylacetate are added in order to carry out an extraction. Then the organicphase is washed with an acidified aqueous solution (HCl 1%). The ethylacetate is evaporated off under reduced pressure. A white powder isobtained with a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass spectrometry (Electrospray®)

Peak detected by mass spectrometry: {M+H}⁺=398

EXAMPLE 5 4,22-cholestadien-3-one oxime (R=R 4)

10 mg of 4,22-cholestadien-3-one (0.026 mmol) is solubilized in 5 ml ofpyridine in a 10 ml flask, then 100 mg of hydroxylamine hydrochloride isadded. Stirring is maintained for 24 hours at ambient temperature, andthe solvent is evaporated off under reduced pressure. Water then ethylacetate are added in order to carry out an extraction. Then the organicphase is washed with an acidified aqueous solution (HCl 1%). The ethylacetate is evaporated off under reduced pressure. A white powder isobtained with a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass spectrometry (Electrospray®)

Peak detected by mass spectrometry: {M+H}⁺=398

EXAMPLE 6 4-stigmasta-ene-3-one oxime (R=R 3)

100 mg of 4-stigmasta-ene-3-one (0.24 mmol) is solubilized in 10 ml ofpyridine in a 50 ml flask, then 100 mg of hydroxylamine hydrochloride isadded. Stirring is maintained for 24 hours at ambient temperature, andthe solvent is evaporated off under reduced pressure. Water then ethylacetate is added in order to carry out an extraction. Then the organicphase is washed with an acidified aqueous solution (HCl 1%). The ethylacetate is evaporated off under reduced pressure. A white powder isobtained with a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass spectrometry (Electrospray®)    -   Peak detected by mass spectrometry: {M+H}⁺=428

EXAMPLE 7 4.6, 22-ergosta-trien-3-one oxime (R=R 6)

100 mg of 4,6,22-ergosta-trien-3-one (0.25 mmol) is solubilized in 10 mlof pyridine in a 50 ml flask, then 100 mg of hydroxylamine hydrochlorideis added. Stirring is maintained for 24 hours at ambient temperature,and the solvent is evaporated off under reduced pressure. Water thenethyl acetate are added in order to carry out an extraction. Then theorganic phase is washed with an acidified aqueous solution (HCl 1%). Theethyl acetate is evaporated off under reduced pressure. A white powderis obtained with a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass spectrometry (Electrospray®)    -   Peak detected by mass spectrometry: {M+H}⁺=410

EXAMPLE 8 1.4, 6-cholesta-trien-3-one oxime (R=R1)

100 mg of 1,4,6-cholesta-trien-3-one (0.26 mmol) is solubilized in 10 mlof pyridine in a 50 ml flask, then 100 mg of hydroxylamine hydrochlorideis added. Stirring is maintained for 24 hours at ambient temperature,and the solvent is evaporated off under reduced pressure. Water thenethyl acetate are added in order to carry out an extraction, then theorganic phase is washed with an acidified aqueous solution (HCl 1%). Theethyl acetate is evaporated off under reduced pressure. A white powderis obtained with a yield greater than 50%.

Analysis

-   -   Liquid chromatography/Mass Spectrometry (Electrospray®)    -   Peak detected by mass spectrometry: {M+H}⁺=396

Pharmacological Study

The following compounds of formula I were tested:

No. 1 Cholest-4-en-3-one oxime described in Gamma irradiation ofcholestenone oximes. Uenseren, Envare. Cekmece Nucl. Res. Train. Cent.,Istanbul, Turk. Avail. INIS. Report (1976), (CNAEM-R-157), 21 pp. From:INIS Atomindex 1977, 8(6), Abstr. No. 295540. 2 Compound of Example 3 3Cholestan-3-one oxime commercially available from Sigma-Aldrich 4Cholest-4,24-dien-3-one described in Chemical synthesis ofcholest-5,7,24-trien-3beta-ol and demonstration of its conversion tocholesterol in the rat. Scallen, Terence J. Univ. of Minnesota,Minneapolis, Biochemical and Biophysical Research Communications (1965),21(2), 149-55. 5 4,22-cholest-dien-3-one described in 3-Keto-Δ4-steroidsfrom 3-hydroxy-ene-4 (or ene-5)- steroids. Yamanaka, Toru; Imai,Takashi. (Takasago Perfumery Co., Ltd., Japan). JP 52116456 19770929Showa. Application: JP 76-32979 19760325. CAN 88: 136852 AN 1978: 1368526 Compound of Example 4 7 Compound of Example 5 8 Compound of Example 69 5-beta-hydroxy-cholestan-3-one commercially available fromSigma-Aldrich 10 5-alpha-hydroxy-cholestan-3-one commercially availablefrom Sigma-Aldrich 11 5-alpha-cholest-6-en-3-one commercially availablefrom Sigma-Aldrich 12 24-methylcholest-4,6,22-trien-3-one described inPreparation of 3-beta-hydroxy-(24R)- methylcholest-5-ene. Khripach, V.A.; Zhabinskii, V. N.; Zhernosek, E. V.; Lakhvich, F. A. (Institute ofBioorganic Chemistry, Academy of Sciences, Belorussian S.S.R., USSR).U.S.S.R. (1990), and SU 1594181 19900923: SU 88-4612746 19881201. CAN114: 102558 AN 1991: 102558 13 Cholest-5-en-3-one oxime-described inGamma irradiation of cholestenone oximes. Uenseren, Envare. CekmeceNucl. Res. Train. Cent., Istanbul, Turk. Avail. INIS. Report (1976),(CNAEM-R-157), 21 pp. 14 Cholest-4,6-dien-3-one oxime described inAzasteroid from cholesta-4,6-dien-3-one. Ahmad, Mohammed S.; Siddiqui,A. H.; Shafiullah; Logani, S. C. Aligarh Muslim Univ., Aligarh, India.Australian Journal of Chemistry (1969), 22(1), 271-4. 15 Compound ofExample 7 16 5-alpha-cholest-1-en-3-one oxime described inPolyphosphoric acid-catalyzed Beckmann rearrangement of 3-keto-steroidoximes. Kobayashi, Masaru; Shimizu, Yuzuru; Mitsuhashi, Hiroshi. Fac.Pharm. Sci., Hokkaido Univ., Sapporo, Japan. Chemical & PharmaceuticalBulletin (1969), 17(6), 1255-60. 17 Compound of Example 8

1. Effects of the Compounds of Formula I on the Survival of the MotorNeurons

In order to demonstrate the neuroprotective action of the compounds offormula I, the applicant studied their activity on an in vitro model oftrophic deprivation of rat motor neurons. Reference can advantageouslybe made to the applicant's patent application WO 0142784 on theculturing of motor neurons of the spinal cord.

The spinal cord of E14 rat embryos is dissected and the ventral part isisolated by trituration after trypsination. The motor neurons areseparated from the other spinal cells by a known method (Camu et al.,1993, Purification of spinal motor neurons from chicken and rat embryosby immunopanning. In <<Immunoselection Strategies for Neural cellculture>>, Neuroprotocols: A companion to Methods in Neurosciences 2,191-199; Henderson et al., 1993, Neutrophins promote motor neuronsurvival and are present in embryonic limb bud. Nature 363(6426):266-70). The cells are centrifuged on a density gradient. Themotor neurons are enriched in the fraction of the large cells (the leastdense). The cells of this fraction are incubated with an anti-p75antibody, a surface antigen present on the motor neurons. Secondaryantibodies coupled with magnetic balls are added and the mixture ofcells is passed through a column in a magnet (Arce et al., 1999). Onlythe motor neurons are retained: their purity is of the order of 90%.

The motor neurons are inoculated at low density in culture wells on asubstrate of polyornithine-laminin in a neurobasal medium supplementedaccording to Raoul et al., 1999, Programmed cell death of embryonicmotor neurons triggered through the Fas death receptor. J Cell Biol147(5):1049-62. Negative controls (absence of trophic factors) andpositive controls (in the presence of BDNF (Brain-Derived NeurotrophicFactor) at 1 ng/ml, GDNF (Glial-Derived Neurotrophic Factor) at 1 ng/mland CNTF (Ciliary Neurotrophic Factor) at 10 ng/ml, marketed by theAmerican company PEPROTECH, Inc. and the Sigma-Aldrich company, areincluded in each series.

The compounds to be tested are added 60 minutes after the inoculationand the cultures are maintained at 37° C. under 5% CO₂ for 3 days.

The motor neurons have a spontaneous tendency to die in the absence ofneurotrophic factors (Pettmann and Henderson, 1998, Neuronal cell death.Neuron 20 (4):633-47). After 3 days, the survival is evaluated by afluorescence measurement after incubation of the cells in the presenceof calcein which becomes fluorescent in the living cells.

After 3 days in culture at 37° C., under 5% CO₂ and in saturatinghumidity, up to 50% of the motor neurons initially inoculated survive inthe medium supplemented with neurotrophic factors, while less than 15%of the motor neurons survive in basal medium alone.

The activity of the compounds to be tested was evaluated according totheir capacity to prevent the death of the motor neurons when they areadded to the neurobasal medium compared with the survival of the motorneurons in medium supplemented with neurotrophic factors.

The compounds of formula I according to the invention have shown anactivity with a concentration capable of allowing a better survival rateof the motor neurons in the basal medium. This survival rate isexpressed by a number, the ratio. If the ratio is greater than 0, theeffect of the compounds on the survival of the motor neurons ispositive.

The results obtained are as follows:

Compound No. Concentration in μM Ratio 1 5 1 2 5 0.6 6 5 0.5 7 6 0.25 810 0.3 15 5 0.2 17 5 0.3 9 1 0.5

Because of their trophic effect on the spinal motor neurons, thecompounds of formula I according to the invention therefore prove to beuseful as a medicament, in particular in the treatment of amyotrophies,in particular in the treatment of amyotrophic lateral sclerosis orinfantile spinal muscular atrophy, and in the treatment of traumas ofthe spinal cord.

2. Effects of the Compounds of Formula Ion Neuroprotection

An axotomy of the facial nerve is performed on new-born young rats of2-3 days old. The animals receive the cholest-4-en-3-one oxime 4 hoursbefore the unilateral section of the nerve then daily for 5 days bysub-cutaneous route. Seven days after the section of the nerve, theanimals are anaesthetized, then fixed by intra-cardial perfusion ofparaformaldehyde. The brain is then removed and placed in paraffin. Thehistological analysis of compound sections of 7 μm of the nucleus of thefacial nerve, stained with cresyl violet, allows the number of motorneurons of the intact side as well as of the side of the nerve which hasbeen sectioned to be counted (Casanovas et al., Prevention bylamotrigine, MK-801 and N omega-nitro-L-arginine methyl ester ofmotoneuron cell death after neonatal axotomy, Neuroscience, 1996, 71,313-325).

The results obtained are as follows:

The survival of the motor neurons of the nucleus of the facial nerve innew-born rats which are axotomized and treated with cholest-4-en-3-oneoxime is increased up to 40% at a dose comprised between 3 and 100mg/kg, according to the administration route, compared with the nervewhich is not sectioned.

3. Effects of the Compounds of Formula Ion Neuroprotection

Crushing of the sciatic nerve is carried out on adult mice according tothe method described by Azzouz et al., Enhancement of mouse sciaticnerve regeneration by the long chain fatty alcohol, N-hexacosanol, Exp.Neurol., 1996, 138: 189-97). The animals receive the compounds to betested the day of the crushing then each day for 4 weeks and bysub-cutaneous route. Each week the animals are anaesthetized in order tomake an electromyographic recording in the calf muscle aftersupramaximal stimulation of the sciatic nerve. The amplitude, theduration and the latency of the action potentials mentioned (CMAP) arethus measured. Four weeks after the crushing of the nerve, the animalsare sacrificed by injection of a lethal dose of anaesthetic and asegment of damaged nerve is sampled, fixed in 4% glutaraldehyde andplaced in resin. The histological analysis of compound sections, stainedwith toluidine blue, allows a semi-automated counting of the degeneratedand non-degenerated fibres, as well as a measurement of the size of theaxons and of the thickness of the myelin sheath of the non-degeneratedfibres.

The results obtained are as follows:

The administration of cholest-4-en-3-one oxime reduces from 20 to 40%the number of degenerated fibres compared with the non-treated animals.In a still more spectacular way, the regeneration of the nerve fibres isgreatly stimulated by the compounds to be tested from the second weekafter the crushing, with a maximum effect observed four weeks after thecrushing. Thus four weeks after the crushing, the amplitude of the CMAPsin the mice treated with the compounds to be tested at a dose between0.3 and 30 mg/kg/day, according to the administration route, isincreased by 40 to 70% and the nerve conduction velocity is improved by30 to 50% compared with the non-treated mice.

4. Effects of the Compounds of Formula I on Protection of StriatalNeurons from Death Induced by the Overexpression of a Mutated Form ofHuntingtin

Primary cultures of striatal neurons are prepared as described in theliterature (Primary striatal neuronal culture, Mao L. et al., MethodsMol. Med., 2003, 79:379-86). The cells undergo electroporation accordingto the procedure described by Raoul et al., (Motoneuron death triggeredby a specific pathway downstream of Fas. potentiation by ALS-linked SOD1mutations Neuron, 2002, 35:1067-83) before inoculation with anexpression vector or plasmid containing a promoter element followed bythe DNA coding for a truncated form of huntingtin which comprises thefirst 480 amino acids and 68 CAG (Saudou et al., huntingtin acts in thenucleus to induce apoptosis but death does not correlate with theformation of intranuclear inclusions, Cell, 1998, 95:55-66). A secondexpression vector containing the DNA coding the green fluorescentprotein (GFP) also undergoes electroporation and serves as a reportergene. The DNA of the plasmid coding the huntingtin was prepared bypurification with cesium chloride. The plasmid containing the GFPsequence was prepared on Qiagen columns. The integrity of the DNAsequences is verified by sequencing, transfection and western blotting.The cells which survive the electroporation are inoculated at a densityof 4000 cells per well of a 96-well plate. The culture takes place inNeurobasal medium (GIBCO) complimented with pyruvate and B-27 (BecktonDickinson). The cells are maintained in culture for 7 days withoutchanging the medium.

The treatments with the compounds to be tested are carried out justafter the inoculation at a final concentration of 1 μM in 0.5% ofdimethyl sulphoxide (DMSO). The positive controls are carried out by theaddition of BDNF at 5 ng/ml final. The negative controls only receive0.5% DMSO.

Cell death is evaluated after the 7 days by counting the number ofliving cells expressing the GFP.

The activity of the compounds to be tested was evaluated by theircapacity to prevent the death of the striatal neurons cultured in theneurobasal medium compared with the survival of the striatal neurons inmedium supplemented with BDNF (Brain-Derived Neurotrophic Factor).

The results obtained are as follows:

Compound No. Concentrations in μM Ratio 1 1 0.3 4 1 0.2 9 1 0.5 11 1 0.312 1 0.3

At the concentration of 10⁻⁶ M, the compounds to be tested demonstratean protective effect against cell death induced by mutated huntingtin ofup to 60 compared with the cells treated with BDNF.

Because of their neuroprotective effect, the compounds of formula Iaccording to the invention thus prove to be useful as medicamentsintended for the treatment or the prevention of neurodegenerativediseases, in particular in the treatment of spinal amyotrophies,amyotrophic lateral sclerosis, in the treatment of traumatisms of thespinal cord and of the peripheral nerves and in the treatment ofHuntington's chorea.

5. Effects of the Compounds of Formula I on the Potentialization of theGABA_(A) Receptors by Steroids

Electrophysiological experiments were carried out in vitro with ratneurons (slices of hippocampus, neurons sensitive in culture), asfollows:

The experiments are carried out with hippocampi removed from rats of theSprague Dawley strain aged from 10 to 21 days. The animals aresacrificed by decapitation, and their brain is rapidly transferred intoACSF (Artificial Cerebro-Spinal Fluid) without sodium (composition: 2 mMKCl, 1.2 mM NaH₂PO₄, 2 mM MgCl₂, 0.5 mM CaCl₂, 26 mM NaHCO₃, 11 mMglucose, 250 mM saccharose), maintained at a temperature of 4° C. andcarbogen (a 95% O₂, 5% CO₂ mixture) is bubbled through. The hippocampiare extracted from each cerebral hemisphere then cut up into slices of300 μM thickness using a “tissue chopper”. The slices are left to restin ACSF medium (composition: 126 mM NaCl, 2 mM KCl, 1.2 mM NaH₂PO₄, 2 mMMgCl₂, 0.5 mM CaCl₂, 26 mM NaHCO₃, 11 mM glucose) at ambienttemperature, at least one hour before recording. Using a pipette, oneslice is then deposited on a multi-electrode recording system (MEA,MultiChannel Systems™, Germany) and positioned so as to cover the 64electrodes. In order to avoid any change in position by the perfusionflow, a grid weighted down by a platinum <<U>> is positioned on theslice. The MEA is then rapidly inserted into the recording system andthe chamber perfused with ACSF with carbogen bubbled through; the middleof the chamber is connected to earth. The perfused ACSF as well as theMEA are maintained at a temperature of 37° C. One of the 60 electrodesis selected as a stimulation electrode. A biphasic stimulation currentwith an intensity of 300 μA over 0.1 ms is generated from this electrodeat a rate of one stimulation every 30 seconds. The responses producedare seen simultaneously at the level of the 59 other electrodes of theMEA. The axons of the Schaeffer collaterals are stimulated in the CA3.In the CA1 region, 6 to 12 electrodes are selected in order to recordthe field potentials which correspond to the sum of the variations ofthe postsynaptic potentials of the neurons situated close to eachelectrode. The molecules tested are dissolved in the ACSF medium (thefinal concentration of DMSO is ≦0.1%) and carried over onto the slice bymeans of the perfusion system.

The sensitive rat neurons are cultured for 1 to 2 days and then recordedwith the patch clamp technique as described in De Roo M. et al.(Dehydroepiandrosterone potentiates native ionotropic ATP receptorscontaining the P2X2 subunit in rat sensory neurones, J Physiol, 2003,552:59-71).

The results obtained are as follows:

On the slices of hippocampus, a small fraction of the postsynaptic fieldpotentials recorded in the CA1 region corresponds to a stimulatingGABA_(A) transmission: it is blocked by standard antagonists of theGABA_(A) receptors (20 μM bicuculline or 50 μM picrotoxin) andpotentialized by positive allosteric modulators of the GABA_(A)receptors: benzodiazepine (1-10 μM of diazepam) and steroids (10-1000 nMallopregnanolone or tetrahydrodeoxycorticosterone). The potentializingeffect of the steroids disappears when the GABA_(A) receptors aredesensitized by the addition of a saturating concentration of GABA (10mM). The stimulating effect of the GABA is linked to the passage of thebicarbonate ions (HCO₃ ⁻) through the GABA_(A) receptors.

In the sensitive neurons recorded with patch-clamp, currents areproduced by selectively opening the GABA_(A) receptors of these neuronsby rapidly applying a GABA_(A)-selective agonist, isoguvacine. Thechlorine currents thus produced are blocked by bucuculline andpotentialized by allopregnanolone.

The results show that cholest-4-en-3-one oxime (tested alone up to 10μM) has no effect on the GABA_(A) receptors (slice of hippocampus orsensitive neurons). By contrast, cholest-4-en-3-one oxime is capable ofblocking the effect of positive allosteric modulators (allopregnanoloneor tetrahydrodeoxycorticosterone) of the GABA_(A) receptors in adose-dependent way and for concentrations equivalent to those whereneurotrophic effects are observed in vitro.

The compounds of formula I therefore have a “protective” effect in allthe pathologies where the overactivation of the GABA_(A) receptors, dueto the presence of allopregnanolone and/ortetrahydrodeoxycorticosterone, can have a harmful effect, either forinhibitory effects or on stimulating effects mediated by the GABA_(A)receptors.

Toxicological Study

The administration to mice, in particular of cholest-4-en-3-one oxime,by oral, sub-cutaneous, intra-peritoneal and intravenous routes, atdoses ranging up to 300 mg/kg/day per treatment with a dailyadministration which can last up to 28 days, did not show significanttoxicity.

In monkeys, the administration by oral route of increasing daily dosesup to 1500 mg/kg over a period of 10 days revealed no toxicity.

1. A method for providing neuroprotection to a patient suffering fromspinal muscular atrophy (SMA), comprising administering to the patient aneuroprotective-effective amount of a compound of formula I

in which X is an oxygen atom or an ═N—OH group, R is selected from thegroup consisting of

A is a hydrogen atom or together with B a carbon-carbon bond, B is ahydrogen atom, a hydroxy group or together with A a carbon-carbon bond,C is a hydrogen atom or together with D a carbon-carbon bond, D is ahydrogen atom or together with C a carbon-carbon bond, E is a hydrogenatom or together with F a carbon-carbon bond, F is a hydrogen atom ortogether with E a carbon-carbon bond, or an addition salt with apharmaceutically acceptable acid.
 2. The method of claim 1, wherein thecompound of formula I is cholest-4-en-3-one oxime.